The de novo biosynthesis of cholesterol in the endoplasmic reticulum proceeds by a multistep process. Acetyl coenzyme A (-CoA) is condensed and converted to 3-hydroxy 3-methylgutaryl (HMG)-CoA. HMG-CoA is reduced to mevalonate by the action of HMG-CoA reductase. In consecutive enzyme-catalyzed reactions, mevalonate is converted to isopentenyl pyrophosphate (IPPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) and squalene. Squalene is oxidized to squalene 2,3-epoxide by squalene epoxidase. Rearrangements of squalene 2,3-epoxide lead to lanosterol. Lanosterol is converted to cholesterol in the endoplasmic reticulum in another series of enzymatic reactions.
HMG-CoA reductase is considered one of the major regulatory enzymes in cholesterol biosynthesis. HMG-CoA reductase inhibitors such as lovastatin are widely used to lower plasma cholesterol levels. Since the HMG-CoA reductase-catalyzed production of mevalonate is an early step in the cholesterol biosynthetic pathway, HMG-CoA reductase inhibition depletes many other intermediates of the cholesterol biosynthetic pathway.
Many intermediates in the cholesterol biosynthetic pathway depleted by inhibition of HMG-CoA reductase have additional roles in cellular function. For instance, FPP and FPP-derived geranylgeranyl pyrophosphate (GGPP) covalently modify proteins, heme, and tRNA, and are precursors for biologically important molecules such as dolichols and ubiquinones (Grunler, J. et al. (1994) Biochim. Biophys. Acta 1212:259-277). Posttranslational protein isoprenylation promotes the anchoring of proteins to membranes and serves as a regulatory signal (Glomset, J. A. et al. (1990) Trends Biochem. Sci. 15:139-142). For example, the Rab proteins are a class of small GTP binding proteins which are involved in intracellular vesicle trafficking. Isoprenylated Rab proteins on the surface of vesicles interact with GTPase-activating proteins and specific Rab receptors on the target membrane, leading to membrane fusion. When cellular isoprenoid synthesis is blocked by lovastatin, Rab proteins that are normally localized in membranes of the endoplasmic reticulum, Golgi apparatus, and endosomes accumulate in the cytosol (Kinsella B. T. et al. (1992) J. Biol. Chem. 267:3940-3945).
Defects in protein isoprenylation caused by depletion of FPP and its metabolites may have undesirable biological consequences. The vesicular trafficking of integral membrane proteins is compromised by altered Rab isoprenylation resulting from depletion of intracellular FPP and GGPP. For instance, cystic fibrosis transmembrane conductance regulator (CFTR) function in a primary human airway epithelial cell line is compromised by lovastatin (Shen, B.-Q. et al. (1995) J Biol. Chem. 270:25102-25106). Lovastatin is proposed to disrupt the trafficking of CFTR to the apical plasma membrane by inhibiting the isoprenylation of Rab or Rab-like trafficking proteins. Protein isoprenylation is also important in the maintenance of retinal cytoarchitecture. Lovastatin produces profound dysplasic-like changes in adult rat retinas primarily in the photoreceptor layer (Pittler, S. J. et al. (1995) J. Cell Biol. 130:431-439). This retinal degeneration is traced to defects in protein isoprenylation.
Arteriosclerosis, a generic term for thickening and hardening of the arterial wall, is responsible for the majority of deaths in the United States and most westernized societies. One type of arteriosclerosis is atherosclerosis, the disorder of the larger arteries that underlies most coronary artery disease, aortic aneurysm, and arterial disease of the lower extremities and also plays a major role in cerebrovascular disease. Atherosclerosis is by far the leading cause of death in the U.S., both above and below age 65 and in both sexes.
A generally accepted theory for the pathogenesis of atherosclerosis is the reaction to injury hypothesis. According to this idea, the endothelial cells lining the intima are exposed to repeated or continuing insults to their integrity. Injury to the endothelium includes metabolic injury, such as chronic hypercholesterolemia. Reduction of hypercholesterolemia results in a decrease in the progression of atherosclerosis in humans and other primates. Drugs that act primarily by lowering low density lipoprotein (LDL) cholesterol are the current drugs of choice for high-risk patients.
It is apparent that, while inhibitors of enzymes at early steps in the cholesterol biosynthetic pathway are effective cholesterol-lowering therapeutics, such inhibitors also deplete other necessary metabolic intermediates. Inhibitors of enzymes which are further along the cholesterol biosynthetic pathway would therefore provide more desirable cholesterol-lowering therapeutics than the inhibitors of earlier pathway enzymes such as HMG-CoA reductase.
The discovery of polynucleotides encoding human squalene epoxidase, and the molecules themselves, presents opportunities to investigate the regulation and control of the later (post-FPP) cholesterol biosynthetic pathway and to elucidate mechanisms for the reduction of hypercholesterolemia in humans. Discovery of molecules related to squalene epoxidase satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the reduction of LDL cholesterol, a key risk factor in atherosclerosis and coronary heart disease.